Diagnosis of systemic lupus erythematosus using oligonucleotides antigens

ABSTRACT

Methods and kits for diagnosing systemic lupus erythematosus (SLE) in a subject are provided. Particularly, the present invention relates to specific oligonucleotide antibody reactivities useful in diagnosing SLE in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/108,889, filed Jun. 29, 2016; which claims priority to PCTInternational Application No. PCT/IL2014/051142 filed Dec. 31, 2014; andto U.S. Provisional Application No. 61/922,114, filed Dec. 31, 2013,which is incorporated herein by reference.

SEQUENCE LISTING

The Sequence Listing submitted herewith is an ASCII text file(2020-01-24_Sequence_Listing_TXT.txt, created on Jan. 24, 2020, 15,484bytes) via EFS-Web is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to oligonucleotide antigens useful indiagnosing an autoimmune disorder such as systemic lupus erythematosus(SLE) in a subject.

BACKGROUND OF THE INVENTION

Systemic lupus erythematosus (SLE), a prototypic autoimmune disease, isassociated with a large spectrum of autoantibodies. IgG antibodies tomore than 100 different antigens including DNA, nucleosomes, histones,viral antigens, transcription factors and more have been reported indifferent SLE patients (Sherer et al., 2004, Semin. Arthritis. Rheum.34:501-37). Surprisingly, there is no serologic diagnosis of SLE and SLEis diagnosed on the basis of eleven criteria defined by the AmericanCollege of Rheumatology (ACR). These criteria include malar rash,discoid rash, photosensitivity, oral ulcers, arthritis, serositis, renaldisorder, neurologic disorder, hematologic disorder (e.g., leucopenia,lymphopenia, hemolytic anemia or thrombocytopenia), immunologic disorderand antibody abnormalities (particularly anti-nuclear antibodies (ANA)and anti-DNA antibodies) (Tan et al., 1997, Arthritis Rheum 1997,40:1725). According to these criteria, subjects can be clinicallydiagnosed with SLE if they meet at least four of the eleven criteria.Recently, the Systemic Lupus Collaborating Clinics (SLICC) revised thesecriteria, as reviewed in Petri et al. (Arthritis and Rheumatism, 2012,Vol. 64, pages 2677-2686). Nevertheless, SLE is still possible even incase when less than four criteria are present.

Although the precise pathology of SLE is not clear, it is widelyaccepted that autoantibodies play an important role. Autoantibodies toDNA are highly heterogeneous with respect to their avidity,immunoglobulin subclass composition, cross-reactivity and complementfixing ability. A number of techniques have been utilized for DNAautoantibodies detection, including immunofluorescent assays (IFA),enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA).However, the clinical value of anti-double stranded DNA (dsDNA)antibodies largely depends on the assay principle and analyticalvariables of the methods used to quantitate and immunologicallycharacterize them.

Park and coworkers (Park et al., “Primary Structures and Chain Dominanceof Anti-DNA Antibodies”, Mol. Cells, 2001, Vol. 11(1), pages 55-63)studied the relative involvement of heavy and light chains of severalanti-DNA autoantibodies in their interaction with several dsDNA targets,amongst which is (GA)₂-(TC)₂ (corresponding to SEQ ID NO: 68 asdescribed herein).

Herkel and coworkers (Herkel et al., “Monoclonal antibody to aDNA-binding domain of p53 mimics charge structure of DNA: anti-idiotypesto the anti-p53 antibody are anti-DNA”, Eur. J. Immunol., 2004, Vol. 34,pages 3623-3632), to some the present inventors, studied twoanti-idiotypic monoclonal antibodies (Idi1 and Idi2) raised againstPAb-421 (a prototypic monoclonal antibody that reacts with theC-terminal DNA-binding domain of p53). These antibodies were found tospecifically recognize both PAb-421 and DNA. In addition, bothantibodies were able to specifically bind single-stranded poly-Gtargets, G20 (corresponding to SEQ ID NO: 43) and T2G16T2 (correspondingto SEQ ID NO: 10 as described herein). However, these antibodies did notbind poly-T, poly-C or poly-A targets.

P. Lenert (“Nucleic acid sensing receptors in systemic lupuserythematosus: development of novel DNA- and/or RNA-like analogues fortreating lupus”, Clinical and Experimental Immunology, 2010, Vol. 161,pages 208-222) reviewed genetic, epigenetic, gender-related andenvironmental factors which are believed to contribute to thepathogenesis of autoimmunity in systemic lupus erythematosus (SLE).Lenert further reviewed several inhibitory oligonucleotides (INH-ODN)aimed to prevent the development of autoimmunity, amongst which is(TTAGGG)₄ (corresponding to present SEQ ID NO: 66).

International Patent Application Publication No. WO 11/099012, to somethe present inventors, relates to methods and kits for diagnosingsystemic lupus erythematosus (SLE) in a subject, using a specificantibody profile. The '012 publication discloses patients having, interalia, increased IgG reactivity to Epstein-Barr Virus (EBV). Additionalpatents and patent applications disclosing diagnosis of autoimmunediseases using a specific antibody profile include WO 10/055510, WO12/052994, US 2005/0260770 and U.S. Pat. No. 8,010,298. Further, USPatent Application Publication No. 2012/0122720 relates to recognizingthe development of cardiovascular disease, e.g., acute myocardialinfarction process in an individual. International Patent ApplicationPublication No. WO 2014/091490, of some the present inventors, relatesto methods for diagnosing SLE or scleroderma by using specific antibodyprofiles against an array of antigens derived from the Epstein-BarrVirus (EBV).

One of the most difficult challenges in clinical management of complexautoimmune diseases such as SLE is the accurate and early identificationof the disease in a patient. There remains a need for improveddiagnostic methods and kits useful in diagnosing SLE in a subject.

SUMMARY OF THE INVENTION

The present invention provides methods and kits for diagnosing anautoimmune disorder, particularly systemic lupus erythematosus (SLE).The present invention further provides antigen probe arrays forpracticing such a diagnosis, and antigen probe sets for generating sucharrays.

The present invention is based, in part, on the unexpected resultsobtained when testing the antibody reactivity of SLE patients comparedto other autoimmune conditions, particularly scleroderma and pemphiguspatients, as well as in comparison to healthy controls. Surprisingly,increased immunoglobulin G (IgG) and IgM reactivities to specificpolynucleotide antigens were found in the tested SLE patients, comparedto healthy controls. Thus, the present invention provides uniqueoligonucleotide antigens, indicative to SLE. The present inventionfurther provides antigen-autoantibody reactivity patterns relevant toSLE. In particular embodiments, the present invention provides highlyspecific, reliable, accurate and discriminatory assays for diagnosingSLE, based on the indicative oligonucleotide antigens, or on reactivitypatterns thereof.

Thus, according to embodiments of the invention, there are providednovel methods for diagnosing and monitoring the progression of SLE.According to embodiments of the invention, the methods comprisedetermining the reactivity of antibodies in a sample obtained or derivedfrom a subject to at least one oligonucleotide antigen as describedherein. The methods of the invention further comprise a step ofcomparing the reactivity of antibodies in the sample to the at least oneoligonucleotide antigen to a control reactivity to said at least oneoligonucleotide antigen. According to certain embodiments, asignificantly higher reactivity of the antibodies in the sample comparedto the reactivity of the healthy control is an indication that thesubject is afflicted with SLE.

Thus, according to a first aspect, the present invention provides amethod of diagnosing systemic lupus erythematosus (SLE) in a subject,the method comprising the steps of obtaining a sample from the subject,determining the reactivity of antibodies in the sample to at least oneoligonucleotide antigen selected from the groups consisting of:GTTTTTTTTTTTTTTTT (SEQ ID NO: 42), TTTTTTTTTTTTTTTTG (SEQ ID NO: 7),GTTTTTTTTTTTTTTTTG (SEQ ID NO: 5), TTTTTTTTTTTTTTTTGG (SEQ ID NO: 28),and TTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 8); or CCATAATTGCAAACGTTCTG (SEQ IDNO: 1) and CCATAATTGCAAAGCTTCTG (SEQ ID NO: 3); or AAAAAAAAAAAAAAAAAAAA(SEQ ID NO: 22); and comparing the reactivity of antibodies in thesample to a reactivity of a healthy control; wherein a significantlyhigher reactivity of the antibodies in the sample compared to thereactivity of the healthy control is an indication that the subject isafflicted with SLE.

As clearly evident, GTTTTTTTTTTTTTTTT (SEQ ID NO: 42), TTTTTTTTTTTTTTTTG(SEQ ID NO: 7), GTTTTTTTTTTTTTTTTG (SEQ ID NO: 5), TTTTTTTTTTTTTTTTGG(SEQ ID NO: 28), and TTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 8) share a commonsequential consensus motif, namely a stretch of at least 16 consecutivethymine nucleotides, preceded or followed by one or two guanosineresidues. As further evident, CCATAATTGCAAACGTTCTG (SEQ ID NO: 1) andCCATAATTGCAAAGCTTCTG (SEQ ID NO: 3) also share a common sequentialconsensus motif, namely CCATAATTGCAAA (SEQ ID NO: 69), followed byeither CGTTCTG (SEQ ID NO: 70) or GCTTCTG (SEQ ID NO: 71).

According to another aspect, the present invention provides a method ofdiagnosing systemic lupus erythematosus (SLE) in a subject, the methodcomprising the steps of obtaining a sample from the subject; determiningthe reactivity of antibodies in the sample to a plurality ofoligonucleotide antigens selected from the group consisting of SEQ IDNOs: 1, 3, 5, 7, 8, 10, 17, 18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65,66 and 67; and comparing the reactivity of antibodies in the sample to areactivity of a healthy control; wherein a significantly higherreactivity of the antibodies in the sample compared to the reactivity ofthe healthy control is an indication that the subject is afflicted withSLE.

In certain embodiments, a significantly higher reactivity of theantibodies in the sample compared to the reactivity of the healthycontrol is an indication that the subject is of increased likelihood tobe afflicted with SLE. In other certain embodiments, where thereactivity of the antibodies in the sample compared to the reactivity ofthe healthy control is not significantly higher, where the reactivity ofthe antibodies in the sample compared to the reactivity of the healthycontrol is the same, where the reactivity of the antibodies in thesample compared to the reactivity of the healthy control is lower orwhere the reactivity of the antibodies in the sample compared to thereactivity of the healthy control is significantly lower, it is anindication that the subject is of decreased likelihood to be afflictedwith SLE. Each possibility represents a separate embodiment of thepresent invention.

In certain embodiments of the methods of the present invention, themethods are preceded by a step comprising obtaining or deriving a samplefrom the subject. In certain embodiments, the sample is obtained orderived from the subject by non-invasive means or methods.

In certain embodiments, determining the reactivity of antibodies in thesample to a plurality of oligonucleotide antigens produces a reactivitypattern, used for the diagnosis of SLE in the subject. Thus, accordingto exemplary embodiments of the invention, the reactivity pattern ofantibodies in the sample to the plurality of oligonucleotide antigens iscompared to the reactivity pattern of antibodies in a samplecorresponding to healthy control subjects to said plurality ofoligonucleotide antigens, wherein a significant difference (typicallyelevation) between the reactivity pattern of the sample and thereactivity pattern of healthy controls indicates that the subject isafflicted with, or in other embodiments has increased likelihood forhaving SLE. Conveniently, the reactivity patterns are calculated andcompared using e.g. learning and pattern recognition algorithms asdescribed herein.

According to some embodiments, the at least one oligonucleotide antigenis selected from the group consisting of SEQ ID NOs: 42, 7, 5, 28 and 8.According to additional embodiments, the at least one oligonucleotideantigen is selected from SEQ ID NOs: 1 or 3. According to additionalembodiments, the at least one oligonucleotide antigen is SEQ ID NO: 22.Each possibility represents a separate embodiment of the invention.

According to another embodiment, the reactivity of antibodies comprisesIgG and IgM reactivities. According to another embodiment, thesignificantly higher reactivity of the antibodies in the samplecomprises increased IgG and/or IgM reactivities. According to anotherembodiment, the increased IgM reactivity is of at least oneoligonucleotide antigen selected from the group consisting of SEQ IDNOs: 42, 7, 5, 28, 8, 1, 3 and 22. According to another embodiment, theincreased IgG reactivity is of at least one oligonucleotide antigenselected from the group consisting of SEQ ID NO: SEQ ID NOs: 42, 7, 5,28, 8, 1, 3 and 22. Each possibility represents a separate embodiment ofthe invention.

In certain embodiments, the subject is positive for antibodies to doublestranded DNA (dsDNA). In other certain embodiments, the subject isnegative for antibodies to dsDNA. Unless otherwise indicated, all singlestranded DNA (ssDNA) and dsDNA samples are calf ssDNA and dsDNA samples.

According to additional embodiments of the methods of the presentinvention, the sample obtained from the subject is a biological fluid.According to some embodiments, the sample is selected from the groupconsisting of plasma, serum, blood, cerebrospinal fluid, synovial fluid,sputum, urine, saliva, tears, lymph specimen, or any other biologicalfluid known in the art. Each possibility represents a separateembodiment of the invention. According to certain embodiments, thesample obtained from the subject is selected from the group consistingof serum, plasma and blood. According to one embodiment, the sample is aserum sample. In certain embodiments, the sample is obtained or derivedfrom the subject by non-invasive means or methods.

According to certain embodiments of the methods of the presentinvention, the control is selected from the group consisting of a samplefrom at least one healthy individual, a panel of control samples from aset of healthy individuals, and a stored set of data from healthyindividuals. Each possibility represents a separate embodiment of theinvention. Typically, a healthy individual is a subject not afflictedwith SLE (or any other form of lupus). In another embodiment, a healthyindividual is a subject not afflicted with an autoimmune disease (e.g.,scleroderma).

According to another embodiment, the method comprises determining thereactivity of antibodies in the sample to a plurality of oligonucleotideantigens.

According to another embodiment, the method comprises determining thereactivity of antibodies in the sample to at least one oligonucleotideantigen selected from the group consisting of SEQ ID NO: 42, SEQ ID NO:7, SEQ ID NO: 5, SEQ ID NO: 28, SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO:3, and SEQ ID NO: 22; and to at least one additional oligonucleotideantigen selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8,10, 17, 18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65, 66 and 67. Eachpossibility represents a separate embodiment of the invention.

According to another embodiment, the method comprises determining thereactivity of antibodies in the sample to at least two oligonucleotideantigens selected from the group consisting of SEQ ID NO: 42, SEQ ID NO:7, SEQ ID NO: 5, SEQ ID NO: 28, SEQ ID NO: 8, SEQ ID NO: 1, SEQ ID NO:3, and SEQ ID NO: 22. Each possibility represents a separate embodimentof the invention.

According to another embodiment, the plurality of antigens is used inthe form of an antigen probe set, an antigen array, or an antigen chip.

According to another aspect, the present invention provides an antigenprobe set comprising a plurality of oligonucleotide antigen probesselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8, 10, 17,18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65, 66 and 67. In anotherembodiment, the antigen probe set comprises the oligonucleotide antigenprobes of SEQ ID NOs: 1, 3, 5, 7, 8, 10, 17, 18, 22, 28, 34, 36, 38, 41,42, 43, 44, 65, 66 and 67.

According to another aspect, the present invention provides an articleof manufacture comprising the antigen probe set described above.

In certain embodiments, the article of manufacture is in the form of anantigen probe array or in the form of an antigen chip or in the form ofa dipstick or in the form of a lateral flow test or in the form of anELISA plate or in the form of a Quanterix system or in the form of adipsticks or any other platform known to those skilled in the art. Incertain embodiments, the article of manufacture is in the form of a kit.

According to certain embodiments, the kit further comprises means fordetermining the reactivity of antibodies in a sample to at least oneantigen of the plurality of antigens. According to another embodiment,the kit further comprises means for comparing reactivity of antibody indifferent samples to at least one antigen of the plurality of antigens.According to another embodiment, the kit further comprises instructionsfor use of the kit for diagnosing SLE.

According to another aspect, there is provided use of the at least oneoligonucleotide antigen selected from the group consisting of SEQ IDNOs: 1, 3, 5, 7, 8, 10, 17, 18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65,66 and 67, for the preparation of a diagnostic kit for diagnosing SLE ina subject. Each possibility represents a separate embodiment of theinvention. The diagnostic kit is, in some embodiments, useful fordetermining the reactivity of antibodies in a sample, therebydetermining the reactivity pattern of the sample to the at least oneoligonucleotide antigen. In some embodiments, a significant difference(e.g., increase) between the reactivity pattern of the sample comparedto a reactivity pattern of a control sample is an indication for SLE.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts individual IgM and IgG reactivities to A20 (SEQ ID NO:22), C20 (SEQ ID NO: 15), G20 (SEQ ID NO: 43) and T20 (SEQ ID NO: 8) insera from healthy subjects (boxes), pemphigus vulgaris (PV) patients(stars), scleroderma (SSc) patients (diamonds), and SLE patients(circles). Subjects were ordered from left to right according to theirreactivity to dsDNA.

FIG. 2 shows IgG reactivity to G20 (SEQ ID NO: 43) compared to all otheroligonucleotides in healthy persons, SSc patients, SLE patients who arenegative or positive for dsDNA. Y axis—reactivities for G20 (SEQ ID NO:43), X axis—reactivities to oligonucleotides. The numbers that appear onboth axes are ×10,000.

FIG. 3 shows mean IgM and IgG binding to polyG and polyToligonucleotides as a function of chain length.

FIG. 4 depicts IgM and IgG reactivities to G17 (SEQ ID NO: 36)oligonucleotide compared to T1G16 (SEQ ID NO: 38) and G16T1 (SEQ ID NO:18).

FIGS. 5A-B depict IgM and IgG reactivities to modified T17oligonucleotides G1T16 (SEQ ID NO: 42) and T16G1 (SEQ ID NO: 7) (5A) andG2T16 (SEQ ID NO: 14) and T16G2 (SEQ ID NO: 28) (5B) compared to T17(SEQ ID NO: 2) reactivities.

FIG. 6 shows IgG and IgM binding to (CG)10 (SEQ ID NO: 25) in healthysubjects, and SSc and SLE patients.

FIGS. 7A-B provide tables of oligonucleotides with increased IgG (7A)and IgM (7B) binding in SLE patients compared to healthy controls. FIG.7AI provides a table of oligonucleotides with increased IgG bindingfound to be extremely SLE-indicative (p value ≤1.87E-08); FIG. 7AIIprovides a table showing that the oligonucleotides of FIG. 7AI aresensitive (≥0.609), specific (≥0.769) and accurate (≥0.687); FIG. 7BIprovides a table of oligonucleotides with increased IgM binding found tobe extremely SLE-indicative (p value ≤1.87E-08); FIG. 7BII provides atable showing that the oligonucleotides of FIG. 7BI are sensitive(≥0.609), specific (≥0.769) and accurate (≥0.687).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of diagnosing an autoimmunedisease or disorder, specifically systemic lupus erythematosus (SLE), ina subject. The present invention further provides antigen probe sets orarrays for practicing such a diagnosis, and identifies specific antigenprobe sets for generating such sets or arrays.

Without wishing to be bound by any particular theory or mechanism ofaction, the invention is based, in part, on the finding of uniqueoligonucleotide antigens highly distinctive between healthy subjects andSLE patients. The invention is further based on the finding that theantibody reactivity profile in serum of SLE patients was clearlydistinct from healthy control individuals. Although serum autoantibodieshave been extensively investigated in SLE, the unique antibody immunesignatures as described herein have not been described before.Advantageously, the unique antibody signatures of the present disclosureprovide highly sensitive and specific assays for diagnosing SLE,particularly of SLE subjects positive to dsDNA.

The present invention provides, in some embodiments, uniqueantigen-autoantibody reactivity patterns particularly relevant to SLE.In the course of investigating anti-DNA autoantibodies, the inventorsexamined the reactivity of IgM and IgG antibodies in the sera of healthypersons and those diagnosed with systemic lupus erythematosus (SLE),scleroderma (SSc), or pemphigus vulgaris (PV) to a variety ofoligonucleotide antigens, using antigen microarray and informaticsanalysis. Surprisingly, all of the human subjects studied, irrespectiveof health or autoimmune disease, manifested relatively high amounts ofIgG antibodies binding to G20 (SEQ ID NO: 43), but not to A20 (SEQ IDNO: 22), C20 (SEQ ID NO: 15), and T20 (SEQ ID NO: 8). Nevertheless, SLEpatients showed increased IgG and IgM reactivities to specificoligonucleotide antigens other than to G20 (SEQ ID NO: 43), which arehighly indicative of SLE, such as GTTTTTTTTTTTTTTTT (SEQ ID NO: 42),TTTTTTTTTTTTTTTTG (SEQ ID NO: 7), GTTTTTTTTTTTTTTTTG (SEQ ID NO: 5),TTTTTTTTTTTTTTTTGG (SEQ ID NO: 28), TTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 8),CCATAATTGCAAACGTTCTG (SEQ ID NO: 1), CCATAATTGCAAAGCTTCTG (SEQ ID NO:3), and AAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 22).

As exemplified herein below, in certain assays, several testedoligonucleotide antigens partly overlapped with the reactivities of calfdouble strand DNA (dsDNA) and single strand DNA (ssDNA). However, asubset of oligonucleotides showed similar or advantageously highersensitivity and/or specificity compared to dsDNA and ssDNA. Theadvantages of using short, single-stranded, synthetic oligonucleotidesequences over oligonucleotides derived from biological sources aremanifold, amongst which fidelity in sequence and ease and cost ofproduction. As noted in the background section herein, the clinicalvalue of anti-dsDNA antibodies largely depends on the assay principleand analytical variables of the methods used to quantitate andimmunologically characterize them. Embodiments of the invention providefor improved assays and methods having advantageous properties forclinical diagnosis compared to hitherto known methods.

The present invention further discloses that SLE patients may beserologically differentiated from scleroderma patients. It is disclosedherein for the first time that SLE subjects have a unique serologicalsignature which can be used to discriminate the subjects from subjectsafflicted with scleroderma. The unique serological signature of SLEsubjects includes increased IgG reactivities as well as decreased IgMreactivities to certain oligonucleotide antigens. Thus, in someembodiments, the present invention provides assays for discriminatingand differentiating between subjects afflicted with SLE and subjectsafflicted with scleroderma.

According to another aspect, the present invention provides a method ofdifferentiating between subjects afflicted with SLE and subjectsafflicted with scleroderma, the method comprising obtaining a samplefrom the subject; determining the reactivity of antibodies in the sampleto at least one oligonucleotide antigen selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 9-15, 17-20, 22-23, 25-34 and 37-39,thereby determining the reactivity pattern of the sample; and comparingthe reactivity pattern of the sample to a control reactivity pattern;wherein a significantly different reactivity pattern of the antibodiesin the sample compared to the control reactivity sample is an indicationthat the subject is afflicted with SLE.

In one embodiment, the reactivity pattern of the sample comprisesincreased IgG reactivity. In particular embodiments, the increased IgGreactivity is of at least one oligonucleotide antigen selected from thegroup consisting of SEQ ID NO: 1, 3, 5, 9-15, 17-20, 22-23, 25-34 and37-39. In another embodiment, the reactivity pattern of the samplecomprises decreased IgM reactivity. In particular embodiments, thedecreased IgM reactivity is of at least one oligonucleotide antigenselected from SEQ ID NO: 25 and 26.

According to some embodiments of the methods for SLE diagnosis in asubject in need thereof, the methods comprises determining thereactivity of antibodies in a sample obtained from the subject to atleast one oligonucleotide antigen selected from the group consisting ofSEQ ID NOs: 42, 7, 5, 28, 8, 1, 3 and 22, or a subset or combinationthereof, thereby determining the reactivity pattern of the sample, anddetermining the subject as a subject afflicted with SLE if thereactivity pattern of the antibodies in the sample is significantlydifferent compared to control.

According to a first aspect, the present invention provides a method ofdiagnosing systemic lupus erythematosus (SLE) in a subject, the methodcomprising the steps of obtaining a sample from the subject, determiningthe reactivity of antibodies in the sample to at least oneoligonucleotide antigen selected from the groups consisting of: SEQ IDNO: 42, SEQ ID NO: 7, SEQ ID NO: 5, SEQ ID NO: 28, and SEQ ID NO: 8; orSEQ ID NO: 1 and SEQ ID NO: 3; or SEQ ID NO: 22; and comparing thereactivity of antibodies in the sample to a reactivity of a healthycontrol; wherein a significantly higher reactivity of the antibodies inthe sample compared to the reactivity of the healthy control is anindication that the subject is afflicted with SLE.

According to a related aspect, the present invention provides a methodof diagnosing systemic lupus erythematosus (SLE) in a subject, themethod comprising the steps of determining the reactivity of antibodiesin a sample obtained from a subject to at least one oligonucleotideantigen selected from the group consisting of: SEQ ID NO: 42, SEQ ID NO:7, SEQ ID NO: 5, SEQ ID NO: 28, and SEQ ID NO: 8; or SEQ ID NO: 1 andSEQ ID NO: 3; or SEQ ID NO: 22; and comparing the reactivity ofantibodies in the sample to a reactivity of a healthy control; wherein asignificantly higher reactivity of the antibodies in the sample comparedto the reactivity of the healthy control is an indication that thesubject is afflicted with SLE.

According to another aspect, the present invention provides a method ofdiagnosing systemic lupus erythematosus (SLE) in a subject, the methodcomprising the steps of obtaining a sample from the subject; determiningthe reactivity of antibodies in the sample to a plurality ofoligonucleotide antigens selected from the group consisting of SEQ IDNOs: 1, 3, 5, 7, 8, 10, 17, 18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65,66 and 67; and comparing the reactivity of antibodies in the sample to areactivity of a healthy control; wherein a significantly higherreactivity of the antibodies in the sample compared to the reactivity ofthe healthy control is an indication that the subject is afflicted withSLE.

According to a related aspect, the present invention provides a methodof diagnosing systemic lupus erythematosus (SLE) in a subject, themethod comprising the steps of: determining the reactivity of antibodiesin a sample obtained from the subject to a plurality of oligonucleotideantigens selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7,8, 10, 17, 18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65, 66 and 67; andcomparing the reactivity of antibodies in the sample to a reactivity ofa healthy control; wherein a significantly higher reactivity of theantibodies in the sample compared to the reactivity of the healthycontrol is an indication that the subject is afflicted with SLE.

The nomenclature used to refer to the oligonucleotide sequence of eacholigonucleotide antigen disclosed in the present invention is asfollows: an oligonucleotide antigen consisting of the oligonucleotidesequence of X₂Y₃Z₂, i.e. two oligonucleotides of X followed by threeoligonucleotides of Y followed by two oligonucleotides of Z is labeledas X2Y3Z2, (X)2(Y)3(Z)2, or XXYYYZZ, or referred to by its correspondingSEQ ID NO.

It should be understood that in this example, X, Y and Z may relate tomore than one oligonucleotide, e.g. to 2-20 oligonucleotides. Therefore,an oligonucleotide antigen consisting of the oligonucleotide sequence ofX₂, wherein X is a stretch of e.g. two oligonucleotides, e.g. YZ, islabeled as X2, (X)2, or YZYZ, or referred to by its corresponding SEQ IDNO.

The terms “systemic lupus erythematosus”, “lupus” and “SLE” as usedherein are interchangeable, and generally refer to an autoimmune diseasecharacterized by the criteria set by the 1982 American College ofRheumatology (ACR) for the diagnosis of SLE, and/or by the SystemicLupus Collaborating Clinics (SLICC) revised criteria, reviewed in Petriet al. (Arthritis and Rheumatism, 2012, Vol. 64, pages 2677-2686).

The terms “oligonucleotide antigen” and “antigen” as used herein areinterchangeable, and generally refer to a stretch of contiguousnucleotides of a certain length. Unless otherwise indicated, the term“oligonucleotide antigen” as used herein relates to a nucleotidesequence of between 15 and 40 nucleotides in length, alternativelybetween 17 and 28 nucleotides in length, or between 18-25 nucleotides inlength. In certain embodiments, an oligonucleotide antigen consists ofat least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 16, or more contiguous nucleotides. Eachpossibility represents a separate embodiment of the invention. Incertain embodiments, an antigen consists of not more than 50, not morethan 45, not more than 40, not more than 35, not more than 30, not morethan 25, not more than 20, not more than 16, or less contiguousnucleotides. Each possibility represents a separate embodiment of theinvention. In certain embodiments, an antigen consists of 10-30, 15-25or 17-20 contiguous nucleotides. In certain embodiments, an antigenconsists of 17, 18, 19 or 20 contiguous nucleotides.

The term “healthy control” as used herein refers to a healthyindividual, a plurality of healthy individuals, a data set or valuecorresponding to or obtained from a healthy individual or a plurality ofhealthy individuals.

The term “sample” as used herein refers to any composition comprising abiological material obtained or derived from a subject. Non-limitingexamples of samples according to the present invention are any kind of abiological tissue or a fluid which comprises antibodies.

As used herein, the “reactivity of antibodies in a sample” or“reactivity of an antibody in a sample” to “an antigen” or to “aplurality of antigens” refers to the immune reactivity of at least oneantibody in the sample to at least one specific antigen selected fromthe plurality of antigens. The immune reactivity of the antibody to theantigen, i.e. its ability to specifically bind the antigen, may be usedto determine the amount of the antibody in the sample. The calculatedlevels of each one of the tested antibodies in the sample arecollectively referred to as the reactivity pattern of the sample tothese antigens. The reactivity pattern of the sample reflects the levelsof each one of the tested antibodies in the sample, thereby providing aquantitative assay. In a preferred embodiment, the antibodies arequantitatively determined.

A “significant difference” between reactivity patterns refers, indifferent embodiments, to a statistically significant difference, or inother embodiments to a significant difference as recognized by a skilledartisan. In yet another preferred embodiment, a significant(quantitative) difference between the reactivity pattern of the sampleobtained from the subject compared to the control reactivity pattern isan indication that the subject is afflicted with SLE. In specificembodiments, up-regulation or higher reactivity of the reactivity of anantibody in a sample to an antigen refers to an increase (i.e.,elevation) of about at least two, about at least three, about at leastfour, or about at least five times higher (i.e., greater) than thereactivity levels of the antibody to the antigen in the control. Inanother embodiment, down-regulation or lower reactivity of thereactivity of an antibody in a sample to an antigen refers to a decrease(i.e., reduction) of about at least two, about at least three, about atleast four, or about at least five times lower than the reactivitylevels of the antibody to the antigen in the control.

In certain embodiments, a subset of oligonucleotide antigen consists ofthe sequence CCATAATTGCAAACGTTCTG (SEQ ID NO: 1), T17 (SEQ ID NO: 2),CCATAATTGCAAAGCTTCTG (SEQ ID NO: 3), G1T16G1 (SEQ ID NO: 5), G10T10 (SEQID NO: 11) and G16T2 (SEQ ID NO: 12), wherein each possibility orcombination thereof represents a separate embodiment of the invention.As exemplified herein below, the subset showed similar or advantageouslyhigher sensitivity compared to dsDNA. In another embodiment, a subset ofoligonucleotide antigen comprises T20 (SEQ ID NO: 8), C20 (SEQ ID NO:15) and A20 (SEQ ID NO: 22), wherein each possibility or combinationthereof represents a separate embodiment of the invention.

According to some embodiments, the at least one oligonucleotide antigenis an oligonucleotide sequence comprising at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous adenine nucleotides.According to another embodiment, the oligonucleotide sequence comprisesat most 20 contiguous adenine nucleotides. According to additionalembodiments, the at least one oligonucleotide antigen is anoligonucleotide sequence comprising at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 contiguous thymine nucleotides.According to another embodiment, the oligonucleotide sequence comprisesat most 20 contiguous thymine nucleotides.

According to additional embodiments, the at least one oligonucleotideantigen is an oligonucleotide sequence comprising at least 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous cytosinenucleotides. According to another embodiment, the oligonucleotidesequence comprises at most 20 contiguous cytosine nucleotides. Accordingto additional embodiments, the at least one oligonucleotide antigen isan oligonucleotide sequence comprising 5-17, 6-17, 7-17, 8-17, 9-17,10-17, 11-17, 12-17, 13-17, 14-17, 15-17, 16-17, or at most 17contiguous guanine nucleotides.

According to some embodiments, the at least one oligonucleotide antigenis selected from the group consisting of SEQ ID NOs: 42, 7, 5, 28 and 8.According to additional embodiments, the at least one oligonucleotideantigen is selected from SEQ ID NOs: 1 or 3. According to additionalembodiments, the at least one oligonucleotide antigen is SEQ ID NO: 22.Each possibility represents a separate embodiment of the invention.

It should be understood each oligonucleotide antigen according to thepresent invention may be bound by IgM antibodies and/or IgG antibodiesfound or isolated from a sample obtained or derived from the testedsubject. Since the relative amounts of IgM antibodies and IgG antibodiesagainst a certain epitope or antigen naturally change over the course oftime, each oligonucleotide antigen according to the present inventionmay be bound by IgM antibodies, IgG antibodies or both. In certainembodiments, the reactivity of antibodies means the reactivity of IgGantibodies. In certain embodiments, the reactivity of antibodies meansthe reactivity of IgM antibodies. According to another embodiment, thesignificantly higher reactivity of the antibodies in the sample meansincreased IgG reactivity. According to another embodiment, thesignificantly higher reactivity of the antibodies in the samplecomprises increased IgM reactivity.

According to another embodiment, the increased IgM reactivity is of atleast one oligonucleotide antigen selected from the group consisting ofSEQ ID NO: 1 (CCATAATTGCAAACGTTCTG), SEQ ID NO: 2 (T17), SEQ ID NO: 3(CCATAATTGCAAAGCTTCTG), SEQ ID NO: 4 (T14), SEQ ID NO: 5 (G1T16G1), SEQID NO: 6 (GACGTT), SEQ ID NO: 7 (T16G1), SEQ ID NO: 8 (T20), SEQ ID NO:9 (T7), and SEQ ID NO: 10 (T2G16T2). According to another embodiment,the increased IgG reactivity is of at least one oligonucleotide antigenselected from the group consisting of SEQ ID NO: 1, 3-5, 8-41.

In certain embodiments, the increased IgM and IgG reactivity is of atleast one oligonucleotide antigen selected from the group consisting ofG1T16 (SEQ ID NO: 42), CCATAATTGCAAACGTTCTG (SEQ ID NO: 1), G1T16G1 (SEQID NO: 5), CCATAATTGCAAAGCTTCTG (SEQ ID NO: 3), A20 (SEQ ID NO: 22) andT20 (SEQ ID NO: 8). In certain embodiments, the increased IgM reactivityis of at least one oligonucleotide antigen selected from the groupconsisting of T16G2 (SEQ ID NO: 28) and T16G1 (SEQ ID NO: 7). Eachpossibility represents a separate embodiment of the invention.

According to another embodiment, the increased IgM reactivity is of atleast one oligonucleotide antigen selected from the group consisting ofSEQ ID NOs: 42, 7, 5, 28, 8, 1, 3 and 22. According to anotherembodiment, the increased IgG reactivity is of at least oneoligonucleotide antigen selected from the group consisting of SEQ ID NO:SEQ ID NOs: 42, 7, 5, 28, 8, 1, 3 and 22. Each possibility represents aseparate embodiment of the present invention.

According to another embodiment, the method further comprisesdetermining the reactivity of the sample to dsDNA and/or ssDNA. Incertain embodiments, the subject is positive for antibodies to dsDNA. Inother certain embodiments, the subject is positive for antibodies tossDNA. In certain embodiments, the subject is negative for antibodies todsDNA. In other certain embodiments, the subject is negative forantibodies to ssDNA. However, as noted in the background section, theclinical value of anti-dsDNA antibodies largely depends on the assayprinciple and analytical variables of the methods used to quantitate andimmunologically characterize them.

It should be understood that in order to perform the methods of thepresent invention, samples obtained or derived from subjects mustcomprise antibodies produced by the subject himself Therefore, samplesmay be obtained or derived from any tissue, organ or liquid naturallycomprising at least a subset of the subject's antibodies. In certainembodiments, the sample obtained from the subject is a biological fluid.According to some embodiments, the sample is selected from the groupconsisting of plasma, serum, blood, cerebrospinal fluid, synovial fluid,sputum, urine, saliva, tears, lymph specimen, or any other biologicalfluid known in the art. Each possibility represents a separateembodiment of the invention. According to certain embodiments, thesample obtained from the subject is selected from the group consistingof serum, plasma and blood. According to one embodiment, the sample is aserum sample. Methods for obtaining and isolating appropriate samplesare well within the purview of the skilled artisan.

According to certain embodiments of the methods of the presentinvention, the control is selected from the group consisting of a samplefrom at least one healthy individual, a panel of control samples from aset of healthy individuals, and a stored set of data from healthyindividuals. Each possibility represents a separate embodiment of theinvention. Typically, a healthy individual is a subject not afflictedwith SLE (or any other form of lupus). In another embodiment, a healthyindividual is a subject not afflicted with any autoimmune disease (e.g.,scleroderma).

In particular embodiments, the significant difference is determinedusing a cutoff of a positive predictive value (PPV) of at least 85%,preferably at least 90%. Determining a PPV for a selected marker (e.g.,an antigen) is well known to the ordinarily skilled artisan and isexemplified in the methods described below. Typically, positivity for anantigen is determined if it detected above 10% of the subjects in aspecific study subgroup using a selected cutoff value, such as PPV ≥90%.For example, antigen i is determined to specifically characterize groupA if it detected at least 10% of the subjects in group A with a PPV ≥90%when compared to a different test group B. Subjects in group A that areabove the cutoff of PPV ≥90% for antigen i are considered to be positivefor antigen i.

An antibody “directed to” an antigen, as used herein is an antibodywhich is capable of specific binding to the antigen. Determining thelevels of antibodies directed to a plurality of antigens includesmeasuring the level of each antibody in the sample, wherein eachantibody is directed to a specific oligonucleotide antigen of theinvention. This step is typically performed using an immunoassay, asdetailed herein.

In other embodiments, determining the reactivity of antibodies in thesample to the at least one antigen (and the levels of each one of thetested antibodies in the sample) is performed by a process comprisingcontacting the sample, under conditions such that a specificantigen-antibody complex may be formed, with at least one antigen (orwhen a plurality of antigens is used, to an antigen probe set comprisingthe plurality of antigens), and quantifying the amount ofantigen-antibody complex formed for each antigen probe. The amount ofantigen-antibody complex is indicative of the level of the testedantibody in the sample (or the reactivity of the sample with theantigen).

In another embodiment the method comprises determining the reactivity ofat least one IgG antibody and at least one IgM antibody in the sample tothe plurality of antigens. In another embodiment, the method comprisesdetermining the reactivity of a plurality of IgG antibodies and at leastone IgM antibody in the sample to the plurality of antigens. In anotherembodiment, the method comprises determining the reactivity of at leastone IgG antibody and a plurality of IgM antibodies in the sample to theplurality of antigens. According to another embodiment, the methodcomprises determining the reactivity of antibodies in the sample to aplurality of oligonucleotide antigens.

Typically, determining the reactivity of antibodies in the sample to atleast one antigen is performed using an immunoassay. Advantageously,when a plurality of antigens is used, the plurality of antigens may beused in the form of an antigen array.

Antigen Probes and Antigen Probe Sets

According to further embodiments, the invention provides antigen probesand antigen probe sets useful for diagnosing SLE, as detailed herein.

The invention further provides a plurality of antigens also referred toherein as antigen probe sets. These antigen probe sets comprise aplurality of antigens which are reactive specifically with the sera ofsubjects having SLE. According to the principles of the invention, theplurality of antigens may advantageously be used in the form of anantigen array. According to some embodiments the antigen array isconveniently arranged in the form of an antigen chip.

A “probe” as used herein means any compound capable of specific bindingto a component. According to one aspect, the present invention providesan antigen probe set comprising a plurality of oligonucleotide antigensselected from the group consisting of: SEQ ID NO:1-67 or anycombinations thereof. According to certain embodiments, the antigenprobe set comprises a subset of the antigens of the present invention.In a particular embodiment, the subset of antigen consists of: SEQ IDNO: 1-10. In another particular embodiment, the subset of antigenconsists of: SEQ ID NO: 1, 3-5 and 8-41. In another embodiment, thesubset of antigen consists of: SEQ ID NO: 1, 2, 3, 5, 11 and 12. In yetanother particular embodiment, the subset of antigen consists of: SEQ IDNO: 8, 15, 22 and 36. In certain embodiment, the subset of antigenconsists of: SEQ ID NO: 42, 7, 5, 28, 8, 1, 3 and 22.

According to another embodiment, the methods of the present inventioncomprise determining the reactivity of antibodies in the sample to atleast one oligonucleotide antigen selected from the group consisting ofSEQ ID NO: 42, SEQ ID NO: 7, SEQ ID NO: 5, SEQ ID NO: 28, SEQ ID NO: 8,SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 22; and to at least oneadditional oligonucleotide antigen selected from the group consisting ofSEQ ID NOs: 1, 3, 5, 7, 8, 10, 17, 18, 22, 28, 34, 36, 38, 41, 42, 43,44, 65, 66 and 67. Each possibility represents a separate embodiment ofthe invention.

According to another embodiment, the methods of the present inventioncomprise determining the reactivity of antibodies in the sample to atleast two oligonucleotide antigens selected from the group consisting ofSEQ ID NO: 42, SEQ ID NO: 7, SEQ ID NO: 5, SEQ ID NO: 28, SEQ ID NO: 8,SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 22. Each possibilityrepresents a separate embodiment of the invention.

The reactivity of antibodies to the plurality of antigens of theinvention may be determined according to techniques known in the art.

Preferably, the plurality of antigens of the methods and kits of theinvention comprises a set of the antigens as disclosed herein. Yet inother embodiments, the plurality of antigens (or the antigen probe set)comprises or consists of a subset thereof, e.g. 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66 or 67 different antigens, each selected from the antigens of thepresent invention, wherein each possibility represents a separateembodiment of the invention. Such subsets may be selected so as toresult in optimal sensitivity and/or specificity of the diagnosticassay.

Antigen probes to be used in the assays of the invention may besynthesized using methods well known in the art.

It should be noted, that the invention utilizes antigen probes as wellas homologs, fragments and derivatives thereof, as long as thesehomologs, fragments and derivatives are immunologically cross-reactivewith these antigen probes. The term “immunologically cross-reactive” asused herein refers to two or more antigens that are specifically boundby the same antibody. The term “homolog” as used herein refers to anoligonucleotide having at least 80%, at least 85% or at least 90%identity to the antigen's nucleic acid sequence. Cross-reactivity can bedetermined by any of a number of immunoassay techniques, such as acompetition assay (measuring the ability of a test antigen tocompetitively inhibit the binding of an antibody to its known antigen).

The term “fragment” as used herein refers to a portion of anoligonucleotide, or oligonucleotide analog which remains immunologicallycross-reactive with the antigen probes, e.g., to immunospecificallyrecognize the target antigen. The fragment may have the length of about80%, about 85%, about 90% or about 95% of the respective antigen.

According to another aspect, the present invention provides an antigenprobe set comprising a plurality of oligonucleotide antigen probesselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8, 10, 17,18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65, 66 and 67.

According to another related aspect, the present invention provides anantigen probe set comprising at least one oligonucleotide antigen probeselected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8, 10, 17,18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65, 66 and 67.

According to another aspect, the present invention provides an articleof manufacture comprising the at least one of the antigen probe setsdescribed above.

In certain embodiments, the article of manufacture is in the form of anantigen probe array or in the form of an antigen chip or in the form ofa dipstick or in the form of a lateral flow test or any other platformknown to those skilled in the art. An “antigen probe array” generallyrefers to a plurality of antigen probes, either mixed in a singlecontainer or arranges in to or more containers. An “antigen chip”generally refers to a substantially two dimensional surface, onto whicha plurality of antigens are attached or adhered. A “dipstick” generallyrefers to an object, onto which a plurality of antigens are attached oradhered, which is dipped into a liquid to perform a chemical test or toprovide a measure of quantity found in the liquid. A “lateral flow test”generally refers to devices intended to detect the presence (or absence)of a target analyte in sample (matrix) without the need for specializedand costly equipment. In certain embodiments, the article of manufactureis in the form of a kit.

According to certain embodiments, the kit further comprises means fordetermining the reactivity of antibodies in a sample to at least oneantigen of the plurality of antigens. According to another embodiment,the kit further comprises means for comparing reactivity of antibody indifferent samples to at least one antigen of the plurality of antigens.According to another embodiment, the kit further comprises instructionsfor use. For example, the aforementioned means may include reagents,detectable labels and/or containers which may be used for measuringspecific binding of antibodies to the antigen probes of the invention.“Means” as used herein may also refer to devices, reagents andchemicals, such as vials, buffers and written protocols or instructions,used to perform biological or chemical assays.

According to another aspect, there is provided use of the at least oneoligonucleotide antigen selected from the group consisting of: SEQ IDNOs: 1, 3, 5, 7, 8, 10, 17, 18, 22, 28, 34, 36, 38, 41, 42, 43, 44, 65,66 and 67, for the preparation of a diagnostic kit for diagnosing SLE ina subject. The diagnostic kit is, in some embodiments, useful fordetermining the reactivity of antibodies in a sample, therebydetermining the reactivity pattern of the sample to the at least oneoligonucleotide antigen. In some embodiments, a significant difference(e.g., increase) between the reactivity pattern of the sample comparedto a reactivity pattern of a control sample is an indication for SLE.

In other embodiments, the plurality of antigens comprised in the antigenprobe set comprises or consists up to 50, 55, 60, 70, 80, 90 or 100different antigens. In other embodiments, the plurality of antigenscomprised in the antigen probe set comprises or consists at least 50,100, 150, 200 or 500 different antigens.

In other aspects, there are provided nucleic-acid vectors comprising theoligonucleotides of the invention and host cells containing them. Thesenucleic acids, vectors and host cells are readily produced byrecombinant methods known in the art. A poly-nucleic acid molecule canalso be produced using recombinant DNA technology (e.g., polymerasechain reaction (PCR) amplification, cloning) or chemical synthesis.Nucleic acid sequences include natural nucleic acid sequences andhomologs thereof, including, but not limited to, natural allelicvariants and modified nucleic acid sequences in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications do not substantially interfere with the nucleicacid molecule's ability to perform the methods of the present invention.

According to the invention, the kits comprise a plurality of antigensalso referred to herein as antigen probe sets. These antigen probe setscomprising a plurality of antigens are reactive specifically with thesera of subjects having SLE. In some embodiments, the antigen probe setscan differentiate between sera of subjects having SLE and subject havingscleroderma. According to the principles of the invention, the pluralityof antigens may advantageously be used in the form of an antigen array.According to some embodiments the antigen array is conveniently arrangedin the form of an antigen chip.

In other embodiments, the kit may further comprise means for determiningthe reactivity of antibodies in a sample to the plurality of antigens.For example, the kit may contain reagents, detectable labels and/orcontainers which may be used for measuring specific binding ofantibodies to the antigen probes of the invention. In a particularembodiment, the kit is in the form of an antigen array.

In some embodiments, the kit comprises means for comparing reactivitypatterns of antibodies in different samples to the plurality ofantigens. In other embodiments, the kit may further comprise negativeand/or positive control samples. For example, a negative control samplemay contain a sample from at least one healthy individual (e.g., anindividual not-afflicted with SLE). A positive control may contain asample from at least one individual afflicted with SLE, or a subtype ofSLE which is being diagnosed. Other non-limiting examples are a panel ofcontrol samples from a set of healthy individuals or diseasedindividuals, or a stored set of data from control individuals.

Antibodies, Samples and Immunoassays

Antibodies, or immunoglobulins, comprise two heavy chains linkedtogether by disulfide bonds and two light chains, each light chain beinglinked to a respective heavy chain by disulfide bonds in a “Y” shapedconfiguration. Each heavy chain has at one end a variable domain (VH)followed by a number of constant domains (CH). Each light chain has avariable domain (VL) at one end and a constant domain (CL) at its otherend, the light chain variable domain being aligned with the variabledomain of the heavy chain and the light chain constant domain beingaligned with the first constant domain of the heavy chain (CH1). Thevariable domains of each pair of light and heavy chains form the antigenbinding site.

The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu)determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM,respectively). The light chain is either of two isotypes (kappa, κ orlambda, λ) found in all antibody classes.

It should be understood that when the terms “antibody” or “antibodies”are used, this is intended to include intact antibodies, such aspolyclonal antibodies or monoclonal antibodies (mAbs), as well asproteolytic fragments thereof such as the Fab or F(ab′)₂ fragments.Further included within the scope of the invention (for example asimmunoassay reagents, as detailed herein) are chimeric antibodies;recombinant and engineered antibodies, and fragments thereof.

Exemplary functional antibody fragments comprising whole or essentiallywhole variable regions of both light and heavy chains are defined asfollows: (i) Fv, defined as a genetically engineered fragment consistingof the variable region of the light chain and the variable region of theheavy chain expressed as two chains; (ii) single-chain Fv (“scFv”), agenetically engineered single-chain molecule including the variableregion of the light chain and the variable region of the heavy chain,linked by a suitable polypeptide linker; (iii) Fab, a fragment of anantibody molecule containing a monovalent antigen-binding portion of anantibody molecule, obtained by treating whole antibody with the enzymepapain to yield the intact light chain and the Fd fragment of the heavychain, which consists of the variable and CH1 domains thereof: (iv)Fab′, a fragment of an antibody molecule containing a monovalentantigen-binding portion of an antibody molecule, obtained by treatingwhole antibody with the enzyme pepsin, followed by reduction (two Fab′fragments are obtained per antibody molecule); and (v) F(ab′)2, afragment of an antibody molecule containing a monovalent antigen-bindingportion of an antibody molecule, obtained by treating whole antibodywith the enzyme pepsin (i.e., a dimer of Fab′ fragments held together bytwo disulfide bonds).

The term “antigen” as used herein is a molecule or a portion of amolecule capable of being bound by an antibody. The antigen is typicallycapable of inducing an animal to produce antibody capable of binding toan epitope of that antigen. An antigen may have one or more epitopes.The specific reaction referred to above is meant to indicate that theantigen will react, in a highly selective manner, with its correspondingantibody and not with the multitude of other antibodies which may beevoked by other antigens. An “antigenic peptide” is a peptide which iscapable of specifically binding an antibody.

In another embodiment, detection of the capacity of an antibody tospecifically bind an antigen probe may be performed by quantifyingspecific antigen-antibody complex formation. The term “specificallybind” as used herein means that the binding of an antibody to a specificantigen probe is not affected by the presence of non-related molecules.

In certain embodiments, the method of the present invention is performedby determining the capacity of an antigen of the invention tospecifically bind antibodies of the IgG isotype, or, in otherembodiments, antibodies of the IgM, isolated from a subject.

Methods for obtaining suitable antibody-containing biological samplesfrom a subject are well within the ability of those of skill in the art.Typically, suitable samples comprise whole blood and products derivedtherefrom, such as plasma and serum. In other embodiments, otherantibody-containing samples may be used, e.g. CSF, urine and salivasamples.

Numerous well known fluid collection methods can be utilized to collectthe biological sample from the subject in order to perform the methodsof the invention.

In accordance with the present invention, any suitable immunoassay canbe used with the subject oligonucleotides. Such techniques are wellknown to the ordinarily skilled artisan and have been described in manystandard immunology manuals and texts. In certain preferableembodiments, determining the capacity of the antibodies to specificallybind the antigen probes is performed using an antigen probe array-basedmethod. Preferably, the array is incubated with suitably diluted serumof the subject so as to allow specific binding between antibodiescontained in the serum and the immobilized antigen probes, washing outunbound serum from the array, incubating the washed array with adetectable label-conjugated ligand of antibodies of the desired isotype,washing out unbound label from the array, and measuring levels of thelabel bound to each antigen probe.

In various embodiments, the method of the present invention furthercomprises diluting the sample before performing the determining step. Inone embodiment, the sample is diluted 1:2, for instance, using PBS. Inanother embodiment, the sample is diluted 1:4, 1:6, 1:8, 1:15, 1:20,1:50, or preferably 1:10. Each possibility represents a separateembodiment of the present invention. In another embodiment, the sampleis diluted in the range of times 2-times 10. In another embodiment, thesample is diluted in the range of times 4-times 10. In anotherembodiment, the sample is diluted in the range of times 6-times 10. Inanother embodiment, the sample is diluted in the range of times 8-times10.

The Antigen Chip

Antigen microarrays are used for the high-throughput characterization ofthe immune response (Robinson et al., 2002, Nat Med 8, 295-301), andhave been used to analyze immune responses in vaccination and inautoimmune disorders (Robinson et al., 2002; Robinson et al., 2003, NatBiotechnol. 21, 1033-9; Quintana et al., 2004; Kanter et al., 2006, NatMed 12, 138-43). It has been hypothesized, that patterns of multiplereactivities may be more revealing than single antigen-antibodyrelationships (Quintana et al., 2006, Lupus 15, 428-30) as shown inprevious analyses of autoimmune repertoires of mice (Quintana et al.,2004; Quintana et al., 2001, J Autoimmun 17, 191-7) and humans (Merbl etal., 2007, J Clin Invest 117, 712-8; Quintana et al., 2003, J Autoimmun21, 65-75) in health and disease. Thus, autoantibody repertoires havethe potential to provide both new insights into the pathogenesis of thedisease and to serve as immune biomarkers (Cohen, 2007, Nat Rev Immunol.7, 569-74) of the disease process.

According to some aspects the methods of the present invention may bepracticed using antigen arrays as disclosed in WO 02/08755 and U.S.2005/0260770 to some of the inventors of the present invention, thecontents of which are incorporated herein by reference. WO 02/08755 isdirected to a system and an article of manufacture for clustering andthereby identifying predefined antigens reactive with undeterminedimmunoglobulins of sera derived from patient subjects in need ofdiagnosis of disease or monitoring of treatment. Further disclosed arediagnostic methods, and systems useful in these methods, employing thestep of clustering a subset of antigens of a plurality of antigens, thesubset of antigens being reactive with a plurality of antibodies beingderived from a plurality of patients, and associating or disassociatingthe antibodies of a subject with the resulting cluster.

U.S. Pat. App. Pub. No. 2005/0260770 to some of the inventors of thepresent invention discloses an antigen array system and diagnostic usesthereof. The application provides a method of diagnosing an immunedisease, particularly diabetes type 1, or a predisposition thereto in asubject, comprising determining a capacity of immunoglobulins of thesubject to specifically bind each antigen probe of an antigen probe set.The teachings of the disclosures are incorporated in their entirety asif fully set forth herein.

In other embodiments, various other immunoassays may be used, including,without limitation, enzyme-linked immunosorbent assay (ELISA), flowcytometry with multiplex beads (such as the system made by Luminex),surface plasmon resonance (SPR), ellipsometry, and various otherimmunoassays which employ, for example, laser scanning, light detecting,photon detecting via a photo-multiplier, photographing with a digitalcamera based system or video system, radiation counting, fluorescencedetecting, electronic, magnetic detecting and any other system thatallows quantitative measurement of antigen-antibody binding.

Various methods have been developed for preparing arrays suitable forthe methods of the present invention. State-of-the-art methods involvesusing a robotic apparatus to apply or “spot” distinct solutionscontaining antigen probes to closely spaced specific addressablelocations on the surface of a planar support, typically a glass support,such as a microscope slide, which is subsequently processed by suitablethermal and/or chemical treatment to attach antigen probes to thesurface of the support. First, the glass surface is activated by achemical treatment that leaves a layer of reactive groups such as epoxygroups on the surface, which bind covalently any molecule containingfree amine or thiol groups. Suitable supports may also include silicon,nitrocellulose, paper, cellulosic supports and the like.

Preferably, each antigen probe, or distinct subset of antigen probes ofthe present invention, which is attached to a specific addressablelocation of the array is attached independently to at least two, morepreferably to at least three separate specific addressable locations ofthe array in order to enable generation of statistically robust data.

According to additional embodiments, the antigen probe set comprises atleast 100, at least 150, at least 200 or more antigens, including one ora plurality of the antigens provided by the present invention. Accordingto additional embodiments, the antigen probe set comprises at least 100,at least 150, at least 200 or more oligonucleotide antigens, includingone or a plurality of the oligonucleotide antigens provided by thepresent invention.

In addition to antigen probes of the invention, the array mayadvantageously include control antigen probes or other standardchemicals. Such control antigen probes may include normalization controlprobes. The signals obtained from the normalization control probesprovide a control for variations in binding conditions, label intensity,“reading” efficiency and other factors that may cause the signal of agiven binding antibody-probe ligand interaction to vary. For example,signals, such as fluorescence intensity, read from all other antigenprobes of the antigen probe array are divided by the signal (e.g.,fluorescence intensity) from the normalization control probes therebynormalizing the measurements. Normalization control probes can be boundto various addressable locations on the antigen probe array to controlfor spatial variation in antibody-ligand probe efficiency. Preferably,normalization control probes are located at the corners or edges of thearray to control for edge effects, as well as in the middle of thearray.

The labeled antibody ligands may be of any of various suitable types ofantibody ligand. Preferably, the antibody ligand is an antibody which iscapable of specifically binding the Fc portion of the antibodies of thesubject used. For example, where the antibodies of the subject are ofthe IgM isotype, the antibody ligand is preferably an antibody capableof specifically binding to the Fc region of IgM antibodies of thesubject.

The ligand of the antibodies of the subject may be conjugated to any ofvarious types of detectable labels. Preferably the label is afluorophore, most preferably Cy3. Alternately, the fluorophore may beany of various fluorophores, including Cy5, Dy5, fluoresceinisothiocyanate (FITC), phycoerythrin (PE), rhodamine, Texas red, and thelike. Suitable fluorophore-conjugated antibodies specific for antibodiesof a specific isotype are widely available from commercial suppliers andmethods of their production are well established.

Antibodies of the subject may be isolated for analysis of their antigenprobe binding capacity in any of various ways, depending on theapplication and purpose. While the subject's antibodies may be suitablyand conveniently in the form of blood serum or plasma or a dilutionthereof (e.g. 1:10 dilution), the antibodies may be subjected to anydesired degree of purification prior to being tested for their capacityto specifically bind antigen probes. The method of the present inventionmay be practiced using whole antibodies of the subject, or antibodyfragments of the subject which comprises an antibody variable region.

Data Analysis

Advantageously, the methods of the invention may employ the use oflearning and pattern recognition analyzers, clustering algorithms andthe like, in order to discriminate between reactivity patterns ofhealthy control subjects to those of patients having SLE. As such, thisterm specifically includes a difference measured by, for example,determining the reactivity of antibodies in a test sample to a pluralityof antigens, and comparing the resulting reactivity pattern to thereactivity patterns of negative and positive control samples (e.g.samples obtained from control subjects which are not afflicted with SLEor patients afflicted with SLE, respectively) using such algorithmsand/or analyzers. The difference may also be measured by comparing thereactivity pattern of the test sample to a predetermined classificationrule obtained in such manner.

In some embodiments, the methods of the invention may employ the use oflearning and pattern recognition analyzers, clustering algorithms andthe like, in order to discriminate between reactivity patterns ofsubjects having a subtype of SLE to control subjects. For example, themethods may include determining the reactivity of antibodies in a testsample to a plurality of antigens, and comparing the resulting patternto the reactivity patterns of negative and positive control samplesusing such algorithms and/or analyzers.

Thus, in another embodiment, a significant difference between thereactivity pattern of a test sample compared to a reactivity pattern ofa control sample, wherein the difference is computed using a learningand pattern recognition algorithm, indicates that the subject isafflicted with SLE. For example, the algorithm may include, withoutlimitation, supervised or non-supervised classifiers includingstatistical algorithms including, but not limited to, principalcomponent analysis (PCA), partial least squares (PLS), multiple linearregression (MLR), principal component regression (PCR), discriminantfunction analysis (DFA) including linear discriminant analysis (LDA),and cluster analysis including nearest neighbor, artificial neuralnetworks, coupled two-way clustering algorithms, multi-layer perceptrons(MLP), generalized regression neural network (GRNN), fuzzy inferencesystems (FIS), self-organizing map (SOM), genetic algorithms (GAS),neuro-fuzzy systems (NFS), adaptive resonance theory (ART).

In certain embodiments, one or more algorithms or computer programs maybe used for comparing the amount of each antibody quantified in the testsample against a predetermined cutoff (or against a number ofpredetermined cutoffs). Alternatively, one or more instructions formanually performing the necessary steps by a human can be provided.

Algorithms for determining and comparing pattern analysis include, butare not limited to, principal component analysis, Fischer linearanalysis, neural network algorithms, genetic algorithms, fuzzy logicpattern recognition, and the like. After analysis is completed, theresulting information can, for example, be displayed on display,transmitted to a host computer, or stored on a storage device forsubsequent retrieval.

Many of the algorithms are neural network based algorithms. A neuralnetwork has an input layer, processing layers and an output layer. Theinformation in a neural network is distributed throughout the processinglayers. The processing layers are made up of nodes that simulate theneurons by the interconnection to their nodes. Similar to statisticalanalysis revealing underlying patterns in a collection of data, neuralnetworks locate consistent patterns in a collection of data, based onpredetermined criteria.

Suitable pattern recognition algorithms include, but are not limited to,principal component analysis (PCA), Fisher linear discriminant analysis(FLDA), soft independent modeling of class analogy (SIMCA), K-nearestneighbors (KNN), neural networks, genetic algorithms, fuzzy logic, andother pattern recognition algorithms. In some embodiments, the Fisherlinear discriminant analysis (FLDA) and canonical discriminant analysis(CDA) as well as combinations thereof are used to compare the outputsignature and the available data from the database.

In other embodiments, principal component analysis is used. Principalcomponent analysis (PCA) involves a mathematical technique thattransforms a number of correlated variables into a smaller number ofuncorrelated variables. The smaller number of uncorrelated variables isknown as principal components. The first principal component oreigenvector accounts for as much of the variability in the data aspossible, and each succeeding component accounts for as much of theremaining variability as possible. The main objective of PCA is toreduce the dimensionality of the data set and to identify new underlyingvariables.

Principal component analysis compares the structure of two or morecovariance matrices in a hierarchical fashion. For instance, one matrixmight be identical to another except that each element of the matrix ismultiplied by a single constant. The matrices are thus proportional toone another. More particularly, the matrices share identicaleigenvectors (or principal components), but their eigenvalues differ bya constant. Another relationship between matrices is that they shareprincipal components in common, but their eigenvalues differ. Themathematical technique used in principal component analysis is calledeigenanalysis. The eigenvector associated with the largest eigenvaluehas the same direction as the first principal component. The eigenvectorassociated with the second largest eigenvalue determines the directionof the second principal component. The sum of the eigenvalues equals thetrace of the square matrix and the maximum number of eigenvectors equalsthe number of rows of this matrix.

In another embodiment, the algorithm is a classifier. One type ofclassifier is created by “training” the algorithm with data from thetraining set and whose performance is evaluated with the test set data.Examples of classifiers used in conjunction with the invention arediscriminant analysis, decision tree analysis, receiver operator curvesor split and score analysis.

The term “decision tree” refers to a classifier with a flow-chart-liketree structure employed for classification. Decision trees consist ofrepeated splits of a data set into subsets. Each split consists of asimple rule applied to one variable, e.g., “if value of “variable 1”larger than “threshold 1”; then go left, else go right”. Accordingly,the given feature space is partitioned into a set of rectangles witheach rectangle assigned to one class.

The terms “test set” or “unknown” or “validation set” refer to a subsetof the entire available data set consisting of those entries notincluded in the training set. Test data is applied to evaluateclassifier performance.

The terms “training set” or “known set” or “reference set” refer to asubset of the respective entire available data set. This subset istypically randomly selected, and is solely used for the purpose ofclassifier construction.

Diagnostic Methods

As used herein the term “diagnosing” or “diagnosis” refers to theprocess of identifying a medical condition or disease (e.g., SLE) by itssigns, symptoms, and in particular from the results of variousdiagnostic procedures, including e.g. detecting the reactivity ofantibodies in a biological sample (e.g. serum) obtained from anindividual, to one or more oligonucleotide antigens. Furthermore, asused herein the term “diagnosing” or “diagnosis” encompasses screeningfor a disease, detecting a presence or a severity of a disease,distinguishing a disease from other diseases including those diseasesthat may feature one or more similar or identical symptoms, providingprognosis of a disease, monitoring disease progression or relapse, aswell as assessment of treatment efficacy and/or relapse of a disease,disorder or condition, as well as selecting a therapy and/or a treatmentfor a disease, optimization of a given therapy for a disease, monitoringthe treatment of a disease, and/or predicting the suitability of atherapy for specific patients or subpopulations or determining theappropriate dosing of a therapeutic product in patients orsubpopulations.

Diagnostic methods differ in their sensitivity and specificity. The“sensitivity” of a diagnostic assay is the percentage of diseasedindividuals who test positive (percent of “true positives”). Diseasedindividuals not detected by the assay are “false negatives.” Subjectswho are not diseased and who test negative in the assay, are termed“true negatives.” The “specificity” of a diagnostic assay is 1 minus thefalse positive rate, where the “false positive” rate is defined as theproportion of those without the disease who test positive. While aparticular diagnostic method may not provide a definitive diagnosis of acondition, it suffices if the method provides a positive indication thataids in diagnosis. The “accuracy” of a diagnostic assay is the proximityof measurement results to the true value. The “p value” of a diagnosticassay is the probability of obtaining the observed sample results (or amore extreme result) when the null hypothesis is actually true.

In certain embodiments, the use of an antigen probe set provided by thepresent invention, or an antigen probe array provided by the presentinvention, results in an antibody reactivity profile which isSLE-indicative (p value ≤1.00E-08), sensitive (≥0.600), specific(≥0.700) and accurate (≥0.600). In certain embodiments, the use resultsin an antibody reactivity profile which is more SLE-indicative (p value≤1.00E-10), sensitive (≥0.700), specific (≥0.800) and accurate (≥0.700).In certain embodiments, the use results in an antibody reactivityprofile which is even more SLE-indicative (p value ≤1.00E-12), sensitive(≥0.800), specific (≥0.900) and accurate (≥0.800). In certainembodiments, the use results in an antibody reactivity profile which isyet even more SLE-indicative (p value ≤1.00E-14), sensitive (≥0.900),specific (≥0.950) and accurate (≥0.900). In certain embodiments, the useresults in an antibody reactivity profile which highly SLE-indicative (pvalue ≤1.00E-16), sensitive (≥0.950), specific (≥0.990) and accurate(≥0.950). Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the oligonucleotide antigens provided by thepresent invention, or the oligonucleotide antigen patterns provided bythe present invention, are SLE-indicative (p value ≤1.87E-08), sensitive(≥0.609), specific (≥0.769) and accurate (≥0.687). In certainembodiments, the oligonucleotide antigens provided by the presentinvention, or the oligonucleotide antigen patterns provided by thepresent invention, are advantageously SLE-indicative (p value≤2.81E-12), sensitive (≥0.657), specific (≥0.798) and accurate (≥0.725).In certain embodiments, the oligonucleotide antigens provided by thepresent invention, or the oligonucleotide antigen patterns provided bythe present invention, are further advantageously SLE-indicative (pvalue ≤8.00E-14), sensitive (≥0.663), specific (≥0.814) and accurate(≥0.738).

In another embodiment, the methods may result in determining a level ofSLE disease activity. In a further embodiment, the methods may result inproviding the comparison to an entity for monitoring SLE diseaseactivity. In these embodiments, the methods can be used, for example, todifferentiate between subjects with active disease and those withnon-active disease.

In some embodiments, the methods of the invention are useful indiagnosing systemic lupus erythematosus (SLE) or lupus. “Lupus” as usedherein is an autoimmune disease or disorder involving antibodies thatattack connective tissue. According to some embodiments, the inventionprovides diagnostic methods useful for the detection of SLE.

In one embodiment, the subject being diagnosed according to the methodsof the invention is symptomatic. In other embodiments, the subject isasymptomatic. In certain embodiments, the subject is not or was notreceiving an immunosuppressive drug or an immunosuppressive treatment.

The diagnostic procedure can be performed in vivo or in vitro,preferably in vitro. In certain embodiments of the methods of thepresent invention, the diagnostic procedure is performed by non-invasivemeans or methods.

Systemic lupus erythematosus (SLE)

The 1982 American College of Rheumatology (ACR) criteria describesfeatures necessary to diagnose SLE. The presence of as few as 4 of the11 criteria yields a sensitivity of 85% and a specificity of 95% forSLE. Patients with SLE may present with any combination of clinicalfeatures and serologic evidence of lupus. The ACR's criteria are (1)Serositis (pleurisy, pericarditis on examination or diagnostic ECG orimaging), (2) Oral ulcers (oral or nasopharyngeal, usually painless;palate is most specific), (3) Arthritis (nonerosive, two or moreperipheral joints with tenderness or swelling), (4) Photosensitivity(unusual skin reaction to light exposure), (5) Blood disorders(leukopenia (<4×10³ cells/μL on more than one occasion), lymphopenia(<1500 cells/μL on more than one occasion), thrombocytopenia (<100×10³cells/μL in the absence of offending medications), hemolytic anemia),(6) Renal involvement (proteinuria (>0.5 g/d or 3+ positive on dipsticktesting) or cellular casts), (7) ANAs (higher titers generally morespecific (>1:160); must be in the absence of medications associated withdrug-induced lupus), (8) Immunologic phenomena (dsDNA; anti-Smith (Sm)antibodies; antiphospholipid antibodies (anticardiolipin immunoglobulinG [IgG] or immunoglobulin M [IgM] or lupus anticoagulant); biologicfalse-positive serologic test results for syphilis, lupus erythematosus(LE) cells (omitted in 1997)), (9) Neurologic disorder (seizures orpsychosis in the absence of other causes), (10) Malar rash (fixederythema over the cheeks and nasal bridge, flat or raised), and (11)Discoid rash (erythematous raised-rimmed lesions with keratotic scalingand follicular plugging, often scarring).

The Systemic Lupus Collaborating Clinics (SLICC) recently revised andvalidated the American College of Rheumatology (ACR) SLE classificationcriteria in order to improve clinical relevance, meet stringentmethodology requirements and incorporate new knowledge in SLE immunology(Petri et al., Arthritis and Rheumatism, 2012, Vol. 64, pages2677-2686). Seventeen criteria were identified, including 11 clinicalcriteria and 6 immunological criteria. The SLICC criteria for SLEclassification requires fulfillment of at least four criteria, with atleast one clinical criterion and one immunologic criterion, or lupusnephritis as the sole clinical criterion in the presence of ANA oranti-dsDNA antibodies.

Two of the most commonly used instruments for SLE diagnosis are theSystemic Lupus Erythematosus Disease Activity Index (SLEDAI) and theSystemic Lupus Activity Measure (SLAM).

The SLEDAI is an index that measures disease activity by weighting theimportance of each organ system involved. The SLEDAI includes 24 items,representing nine organ systems. The variables are obtained by history,physical examination and laboratory assessment. Each item is weightedfrom 1 to 8 based on the significance of the organ involved. Forexample, mouth ulcers are scored as 2, while seizures are scored as 8.The laboratory parameters that are included in the SLEDAI include whiteblood cell count, platelet count, urinalysis, serum C3, C4 andanti-dsDNA. The total maximum score is 105.

The SLAM includes 32 items representing 11 organ systems. The items arescored not only as present/absent, but graded on a scale of 1 to 3 basedon severity. The total possible score for the SLAM is 86. Both theSLEDAI and the SLAM have been shown to be valid, reliable, and sensitiveto change over time (Liang et al. 1989, Arth Rheum 32:1107-18), and arewidely used in research protocols and clinical trials. These indices areparticularly useful for examining the value of newly proposed serologicor inflammatory markers of disease activity in SLE.

Despite the obvious utility of these instruments, there are somedrawbacks. First, there is not always complete agreement between theSLAM and the SLEDAI in the same set of patients. There are severalpossible reasons for these discrepancies. Unlike the SLEDAI, the SLAMincludes constitutional symptoms such as fatigue and fever, which may ormay not be considered attributable to active SLE; this activity indexrelies on physician interpretation. In addition, the SLEDAI does notcapture mild degrees of activity in some organ systems and does not havedescriptors for several types of activity, such as hemolytic anemia.

Scleroderma (or systemic sclerosis) is an autoimmune disease that ischaracterized by endothelial cell damage, fibroblast activation,extracellular matrix (ECM) accumulation and abnormal angiogenesis thatcarries a high rate of morbidity and mortality. One of the major causesof mortality is fibrosis of lung tissue (interstitial lung disease) andsevere pulmonary hypertension. The pathogenesis of scleroderma remainsunclear, but is thought to involve an autoimmune response against targetorgans with early production of autoantibodies and inflammatorymononuclear cell infiltrates followed by loss of organ function andfibrosis. Principal target organs are the skin, the gastrointestinaltract, the lungs and kidneys, although other organs are also frequentlyinvolved. Widespread scleroderma can occur with other autoimmunediseases, including SLE.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

EXAMPLES Materials and Methods

Human Subjects

The study was approved by the Institutional Review Boards of eachparticipating clinical unit; informed consent was obtained from allparticipants. In an initial study, sera derived from blood samplesobtained from 22 healthy subjects, 18 Pemphigus Vulgaris (PV) patients,15 Scleroderma and Systemic Sclerosis (SSc) patients, and 34 SystemicLupus Erythematosus (SLE) patients were tested using an antigenmicroarray that included A20 (SEQ ID NO: 22), C20 (SEQ ID NO: 15), G20(SEQ ID NO: 43) and T20 (SEQ ID NO: 8) single-stranded oligonucleotides.In a follow-up study, sera samples obtained from 23 healthy subjects, 24SSc patients, and 49 SLE patients were tested using an antigenmicroarray that included 58 single-stranded oligonucleotides. Overall,60 SLE patients, 26 SSc patients, 18 PV patients, and 31 healthysubjects were tested. SLE and SSc patients were diagnosed according toclinically accepted criteria (Criteria published by EM Tan et al.Arthritis Rheum 1982; 25:1271, updated by MC Hochberg, Arthritis Rheum1997; 40:1725; Preliminary criteria for the classification of systemicsclerosis (scleroderma). Subcommittee for scleroderma criteria of theAmerican Rheumatism Association Diagnostic and Therapeutic CriteriaCommittee. Arthritis Rheum. 1980; 23(5):581-90). The diagnosis of PV wasbased upon clinical features and laboratory tests: suprabasal separationon histology of skin lesions, positive direct and indirectimmunofluorescence microscopy, and/or ELISA detection of anti-desmogleinAbs (Zagorodniuk I, et al. Int J Dermatol. 2005 July; 44(7):541-4).

Blood samples and clinical data were collected from patients arriving atthe Rheumatology and Nephrology Units at Rabin Medical Center,PetachTikva, Israel; the Rheumatology Unit and the Hematology Departmentof the Sheba Medical Center, Israel; the Department of Dermatology, TelAviv Sourasky Medical Center; and the Dipartimento di Scienze Mediche eChirurgiche, Sezione di Clinica Medica, Polo Didattico, Ancona, Italy.Inclusion criteria were ACR criteria score of >3 at time of diagnosis.Healthy control samples were obtained under study protocols approved bythe Institutional Review Boards of each participating clinical unit;informed consent was obtained from all participants.

Samples were also obtained from 83 healthy subjects of an average age of35, including 47 Africans, 15 White & Caucasian, 4 Indian/Asian/middleeastern and 17 Hispanic; and 77 SLE patients, of the same average age,including 47 Africans, 6 White & Caucasian, 17 Hispanic and 1 of anotherdescent.

Antigens and Serum Testing

In a follow-up study, 58 different oligonucleotides, as well as doubleand single stranded DNA, in various lengths (104 different preparationsoverall), were spotted on epoxy-activated glass substrates (ArrayItSuperEpoxi microarray substrate slides, Sunnyvale, CA). Theoligonucleotides were purchased from SBS Genetech Co., Ltd. (Shanghai,China). The microarrays were then blocked for 1 hour at 37° with 1%bovine serum albumin. Test serum samples in 1% Bovine Serum Albumin(BSA) blocking buffer (1:10 dilution) were incubated under a coverslipfor 1 hour at 37°. The arrays were then washed and incubated for 1 hourat 370 with a 1:500 dilution of two detection antibodies, mixedtogether: a goat anti-human IgG Cy3-conjugated antibody, and a goatanti-human IgM Cy5-conjugated antibody (both purchased from JacksonImmunoResearch Laboratories Inc., West Grove, PA). Image acquisition wasperformed by laser (Agilent Technologies, Santa Clara, CA) and theresults were analyzed using Quantarray software (Packard BioChipTechnologies, Billerica, MA). The quantitative range of signal intensityof binding to each antigen spot was 0-65,000; this range of detectionmade it possible to obtain reliable data at a 1:10 dilution of testserum samples.

Alternatively, oligonucleotide antigens and double and single strandedDNA in various chain lengths were spotted on epoxyhexyltriethoxysilane(EHTES) activated slides. The microarrays were then blocked for 1 hourat room temperature with 1% casein. Test serum samples in 1% caseinblocking buffer (1:20 dilution) were incubated under a coverslip for 1hour at 37°. The arrays were then washed and incubated for 1 hour at 37°with a 1:500 dilution of two detection antibodies, mixed together: agoat anti-human IgG Cy3-conjugated antibody, and a goat anti-human IgMAF647-conjugated antibody (both purchased from Jackson ImmunoResearchLaboratories Inc., West Grove, PA). Image acquisition was performed bylaser at two wavelengths 530 nm and 630 nm (Agilent Technologies, SantaClara, CA) and the results were analyzed using Genepix Pro 7.0 softwarewith default settings. The quantitative range of signal intensity ofbinding to each antigen spot was 0-65,000; this range of detection madeit possible to obtain reliable data at a 1:20 dilution of test samples.

Image Analysis and Data Processing

Each spot's intensity is represented by its pixels' mean aftersubtraction of its local background median, followed by Log 2 transform.Negative spots (following background subtraction) are imputed withbackground-like intensity. The foreground and background intensities ofmultiple spots of each antigen were averaged, and the difference betweenthe foreground and the background was calculated. The resulting valuewas taken as the antigen reactivity of the antibodies binding to thatspotted antigen. All antigens showed meaningful reactivity in asignificant number of slides; thus no antigen was excluded.

Statistical Analysis of Antibody Results

Antigens whose reactivity was higher or lower in a specific studysubgroup compared to other subgroups were identified. Antigens thatallowed for setting a classification threshold such as positivepredictive value (PPV) ≥90% and sensitivity ≥20% were achieved anddetermined to significantly characterize a specific subgroup. SLEpatients were marked positive for dsDNA if their reactivity to dsDNApassed this requirement. For added restriction, only antigens whose pvalue for a two sided t-test (after Benjamini-Hochberg correction formultiple hypothesis) was smaller than 0.05 were selected.

Example 1 Antibodies' Binding to Homo-Nucleotide 20-Mers

Sera samples from healthy subjects, PV, SSc and SLE patients were testedfor binding of serum IgG and IgM antibodies to four 20-merhomo-nucleotides: G20 (SEQ ID NO: 43), A20 (SEQ ID NO: 22), C20 (SEQ IDNO: 15), and T20 (SEQ ID NO: 8) (FIG. 1 ). The reactivities were orderedby each subject's reactivity to dsDNA, from left to right. It can beseen that IgG reactivities to G20 (SEQ ID NO: 43) were very high in allsubjects, and significantly higher than the very low reactivities to theother oligonucleotides. However, PV patients were found to havesignificantly lower IgG and IgM reactivities to G₂₀ than did SScpatients. Apart from that, no difference was found between the studygroups.

IgG reactivities to A20 (SEQ ID NO: 22), C20 (SEQ ID NO: 15), and T20(SEQ ID NO: 8) in SLE patients correlated with their reactivities todsDNA; Patients with low reactivities to dsDNA did not manifestreactivities to A20 (SEQ ID NO: 22), C20 (SEQ ID NO: 15), and T20 (SEQID NO: 8), but several patients with higher reactivities to dsDNA showedsome reactivities to A20 (SEQ ID NO: 22), C20 (SEQ ID NO: 15), and T20(SEQ ID NO: 8) (FIG. 1 ). Healthy subjects, SSc and PV patients had verylow or no reactivities to A20 (SEQ ID NO: 22), C20 (SEQ ID NO: 15), andT20 (SEQ ID NO: 8).

IgM reactivities to the four homo-nucleotide sequences were morediffuse: some subjects in each group showed high reactivities to G20(SEQ ID NO: 43), but, in contrast to the IgG reactivities to G20 (SEQ IDNO: 43), some of the sera showed little or no IgM binding to G20 (SEQ IDNO: 43).

To further characterize the antibodies' reactivity against polyG andpolyT oligonucleotides, an additional study on 23 healthy subjects, 24SSc patients, and 49 SLE patients was performed. An extended microarrayantigen chip was used. The chip contained 58 oligonucleotides, includingpolyG and polyT sequences with and without modifications (see below).The SLE patients were divided according to their reactivity to dsDNA inorder to see if dsDNA positivity or negativity was associated withantibodies to the synthetic oligonucleotides. The results of the IgM andIgG reactivities to G20 (SEQ ID NO: 43), A20 (SEQ ID NO: 22), C20 (SEQID NO: 15), and T20 (SEQ ID NO: 8) of the first study were confirmed.Combining both studies yielded a total of 60 SLE patients, 26 SScpatients, 18 PV patients and 31 healthy subjects who all displayed highIgG reactivities to G20 (SEQ ID NO: 43) and relatively low reactivitiesto A20 (SEQ ID NO: 22), C20 (SEQ ID NO: 15), and T20 (SEQ ID NO: 8).Mean IgG reactivities to G20 (SEQ ID NO: 43) where significantly higherthan the other poly-nucleotides in all the study groups.

The IgG reactivities to G20 (SEQ ID NO: 43) were compared to IgGreactivities to the other oligonucleotides and to ssDNA and dsDNA. FIG.2 shows a scatter plot in which each dot represents the IgG reactivityto G20 (SEQ ID NO: 43) divided by a specific oligonucleotide. Since someof the oligonucleotides and ssDNA and dsDNA were in replicates andmixtures, each subject is represented by 97 spots. Higher reactivity toG20 (SEQ ID NO: 43) compared to a different oligonucleotide would berepresented by a dot above the diagonal. Note that out of the 2231 spotsof the 23 healthy subjects, only 10 were below the diagonal. This numberincreases a little for SSc patients and dsDNA-negative SLE patients, andpeaks for dsDNA-positive patients (FIG. 2 ). Nevertheless, the averageIgG reactivities to G20 (SEQ ID NO: 43) were significantly higher thanIgG reactivities to any other oligonucleotide in all four subgroupstested.

The list of oligonucleotides tested on the antigen chip was as follows:A20 (SEQ ID NO: 22), C20 (SEQ ID NO: 15), T2G16T2 (SEQ ID NO: 10),G2T16G2 (SEQ ID NO: 16), (GA)10 (SEQ ID NO: 44), (GT)10 (SEQ ID NO: 45),G4-7,9,11,14,17,20 (SEQ ID NOs: 46, 37, 13, 17, 34, 47, 41, 36 and 43,respectively), T4-7,9,11,14,17,20 (SEQ ID NOs: 48, 49, 35, 9, 50, 40, 4,2 and 8, respectively), (CG)2-6,8,10 (SEQ ID NOs: 51, 52, 53, 54, 29, 55and 25, respectively), (C*G)2-6,8,10 (*=with C methyl) (SEQ ID NOs: 56,57, 39, 58, 30, 33 and 26, respectively), T1G16T1 (SEQ ID NO: 20), G16T1(SEQ ID NO: 18), T1G16 (SEQ ID NO: 38), G16T2 (SEQ ID NO: 12), T2G16(SEQ ID NO: 24), G1T16G1 (SEQ ID NO: 5), T16G1 (SEQ ID NO: 7), G1T16(SEQ ID NO: 42), T16G2 (SEQ ID NO: 28), G2T16 (SEQ ID NO: 14), GACGCT(SEQ ID NO: 59), GACGTT (SEQ ID NO: 6), G10GACGCT (SEQ ID NO: 27),G10GACGTT (SEQ ID NO: 60), GAGCCT (SEQ ID NO: 21), GAGCTT (SEQ ID NO:61), G10GAGCCT (SEQ ID NO: 19), G10GAGCTT (SEQ ID NO: 62), CCCGGA (SEQID NO: 63), G10CCCGGA (SEQ ID NO: 32), and TCCATAACGTTGCAACGTTCTG (SEQID NO: 64).

Example 2 Antibody Binding to Poly-G or Poly-T is Related to the Lengthof the Homo-Nucleotide

FIG. 3 shows the effect of variable lengths of the nucleotide oligomerson the mean IgG and IgM binding of each tested group to the T or Ghomo-nucleotides. It can be seen that, except for SLE patients positivefor anti-dsDNA who showed reactivities to T₂₀, none of the other groupsshowed appreciable IgG or IgM mean reactivities to any of the poly-Thomo-nucleotides. In contrast, mean IgG reactivities to poly-G in all ofthe sera were high to G20 (SEQ ID NO: 43) and fell significantly as thelengths of the nucleotide chains were reduced to G17 (SEQ ID NO: 36) andbelow. Surprisingly, SLE patients positive for anti-dsDNA manifestedhigher mean IgG reactivities to the shorter G polymers than did theother groups.

Mean IgM binding to G20 (SEQ ID NO: 43) was lower than the IgG binding,and IgM binding was also affected by shortening the length of theoligomer. Note that the mean IgM binding of the SLE patients positive todsDNA did not differ from that of the other groups.

Example 3 The Effects of Adding a Single T to Either the 5′ or the 3′Termini of G₁₆

The degree of binding of IgG or IgM to G17 (SEQ ID NO: 36) compared toG₁₆ to which a single T had been added either at the 5′ or 3′ end of theG-oligonucleotide chain was tested. FIG. 4 shows the results forindividual subjects. It can be seen that both the IgG and IgM binding toT1G16 (SEQ ID NO: 38) was essentially equal to the binding to the G17(SEQ ID NO: 36) chain, as evident from the diagonal between G17 (SEQ IDNO: 36) and T1G16 (SEQ ID NO: 38). However, the binding of each subjectto G16T1 (SEQ ID NO: 18) was considerably less than the binding to G17(SEQ ID NO: 36); a diagonal relationship was no longer present. Thus, itwould appear that the reactivities to poly-G in each of the subjectgroups was highly influenced by the addition of a single T moiety to the3′ end of the poly-G chain but not by the addition of a T to the 5′ endof the G chain; the spatial order of the nucleotides would appear toform an antigen structure critical to antibody binding.

Example 4 The Effects of Single G Additions to the Ends of Poly-TSequences

In view of the marked effects of adding a single T to the 3′ end of apoly-G chain (Example 3), the effects on IgG or IgM antibody binding byadding a single G to either the 5′ or 3′ end of a poly-T chain wastested. FIG. 5A shows that although both IgM and IgG reactivities toG1T16 (SEQ ID NO: 42) and to T16G1 (SEQ ID NO: 7) increased in almostall the subjects (most points are above the diagonal), the increaseswere much more pronounced when the guanine was added to the 5′ end.Similarly IgM and IgG reactivities to G2T16 (SEQ ID NO: 14) and T16G2(SEQ ID NO: 28) were also increased compared to T17 (SEQ ID NO: 2) (FIG.5B).

In summary, reactivities to poly-T oligonucleotides could be increasedsignificantly by the addition of even a single G to either end of thechain; this was in marked contrast to the inhibition of antibody bindingto poly-G by the addition of a single T to the 3′ end of the chain.

Example 5 Reactivities to CpG Repeats

IgM reactivities were measured in three subgroups to a 20-mer formed by10 repetitions of the C-G di-nucleotides, (CG)10 (SEQ ID NO: 25). IgMreactivities to (CG)10 (SEQ ID NO: 25) were high in all but one of thehealthy subjects, in all of the SSc patients and in most of the SLEpatients. Indeed, a subgroup of SLE patients manifested low IgMreactivities to (CG)10 (SEQ ID NO: 25), a significant difference fromthe SSc patients (FIG. 6 ). A group of SLE patients, mainly thosepositive for anti-dsDNA, manifested high IgG reactivities to (CG)10 (SEQID NO: 25).

Example 6 IgG and IgM Reactivities to dsDNA, ssDNA and SyntheticOligonucleotides in SLE Patients Compared to Those of Healthy Subjectsand SSc Patients

Table 1 lists the oligonucleotides with increased or decreased antibodybinding in SLE patients compared to healthy controls or SSc patients. Abroad spectrum of oligonucleotides was found to bind both IgM and IgGantibodies in SLE patients compared to healthy subjects. IgM and IgGreactivities to dsDNA overlapped; 18 of 23 (78%) SLE patients positivefor IgM anti-dsDNA were also positive for IgG anti-dsDNA. Furthermore,the increased IgM and IgG reactivity to the oligonucleotides in the SLEpatients overlapped with the IgM and IgG reactivity to dsDNA,respectively. Thus, the subgroup of dsDNA-positive SLE patients showsincreased reactivities to oligonucleotides generally, perhaps to abackbone structure, compared to the dsDNA-negative patients.

IgG reactivities as well as IgM reactivities to oligonucleotides werefound to be significantly increased in SLE compared to healthy controlsand/or SSc patients.

TABLE 1Antibody reactivities to oligonucleotides in SLE patients compared tohealthy controls and SSc patients. Sensitivity(%) for PPV^(a) ≥ 90%SEQ ID SLE compared to SLE compared to Oligonucleotides NO: controls SScIgM increase dsDNA 47 NS^(b) CCATAATTGCAAACGTTCTG 1 47 NS T₁₇ 2 45 NSCCATAATTGCAAAGCTTCTG 3 43 NS ssDNA 39 NS T₁₄ 4 29 NS G₁T₁₆G₁ 5 24 NSGACGTT 6 24 NS T₁₆G₁ 7 24 NS T₂₀ 8 24 NS T₇ 9 22 NS T₂G₁₆T₂ 10 20 NSIgM decrease (C*G)₁₀ 26 NS 29 (CG)₁₀ 25 NS 27 IgG increase G₁₀T₁₀ 11 78NS CCATAATTGCAAAGCTTCTG 3 71 61 CCATAATTGCAAACGTTCTG 1 69 47 ssDNA 69 39G₁₆T₂ 12 65 39 G₁T₁₆G₁ 5 65 22 dsDNA 65 63 G₆ 13 63 47 G₂T₁₆ 14 63 33C₂₀ 15 61 41 G₂T₁₆G₂ 16 61 NS G₇ 17 61 39 G₁₆T₁ 18 59 22 T₂G₁₆T₂ 10 5947 G₁₀GAGCCT 19 57 41 T₁G₁₆T₁ 20 57 22 GAGCCT 21 51 NS A₂₀ 22 47 39 C₃G₃23 47 35 T₁₄ 4 47 NS T₂G₁₆ 24 47 NS (CG)₁₀ 25 45 31 (C*G)₁₀ 26 45 29G₁₀GACGCT 27 45 45 T₁₆G₂ 28 45 29 (CG)₆ 29 43 31 (C*G)₆ 30 41 33 G₁₀A₁₀31 41 47 G₁₀CCCGGA 32 41 39 (C*G)₈ 33 39 31 T₂₀ 8 37 NS G₉ 34 35 27 T₆35 35 NS G₁₇ 36 31 NS G₅ 37 31 61 T₁G₁₆ 38 27 27 (C*G)₄ 39 24 24 T₁₁ 4024 NS G₁₄ 41 20 NS T₇ 9 20 20 ^(a)PPV, Positive predictive value;^(b)NS, Not significant.

Example 7 IgG and IgM Reactivities to Synthetic Oligonucleotides in SLEPatients Compared to Those of Healthy Subjects

Table 2 lists the oligonucleotides with increased or decreased antibodybinding in two subgroups of subjects (77 SLE patients compared to 83healthy controls).

Mean differences in binding of IgM and IgG antibodies from SLE patientscompared to healthy controls to a variety of oligonucleotide antigensare presented as mean difference, difference significance (p value), andfalse discovery rate-corrected p value (used to correct for multiplecomparisons), wherein positive values indicate an increase in bindingover the level measured in healthy controls (HC) and negative valuesindicate a decrease in binding over the level measured in HC.

A broad spectrum of oligonucleotides was found to bind more IgM and IgGantibodies in SLE patients compared to healthy subjects. Examples ofsuch oligonucleotide antigens are G1T16 (SEQ ID NO: 42),CCATAATTGCAAACGTTCTG (SEQ ID NO: 1), G1T16G1 (SEQ ID NO: 5),CCATAATTGCAAAGCTTCTG (SEQ ID NO: 3), A20 (SEQ ID NO: 22) and T20 (SEQ IDNO: 8). Other oligonucleotides were found to bind more IgM or more IgGantibodies in SLE patients compared to healthy subjects. Examples ofoligonucleotide antigens found to bind more IgM are T16G2 (SEQ ID NO:28) T16G1 (SEQ ID NO: 7).

TABLE 2Reactivities to oligonucleotides in SLE patients compared to controls.IgM IgG IgG Mean IgM Mean FDR^(a) Differ- FDR^(a) Differ- cor- encecorrected ence rected (SLE- IgM p p (SLE- IgG p p Antigen HC) valuevalue HC) value value G₁T₁₆ 0.71558 5.04E−06 3.87E−05 2.132 9.18E−084.22E−06 CCATAATTGCAAACG 0.90735 0.00043208 0.0010001 1.8404 2.08E−074.79E−06 TTCTG G₃₀ 0.85963 0.00079088 0.0015158 1.8957 4.27E−07 6.55E−06G₂₀ 0.50976 0.019515 0.027203 1.8547 1.49E−06 1.71E−05 G₁T₁₆G₁ 0.749970.00045524 0.0010001 1.6919 3.17E−06 2.92E−05 CCATAATTGCAAAGC 0.885580.00044727 0.0010001 1.5353 6.90E−06 4.53E−05 TTCTG (TTAGGG)₄ 0.757094.70E−05 0.00024047 1.3635 2.29E−05 0.00013154 G₁₇ 0.45068 0.0363830.049224 1.4023 6.98E−05 0.00026755 T₂G₁₆T₂ 0.73236 0.0046818 0.00769161.6893 6.68E−05 0.00026755 A₂₀ 0.5841 0.005488 0.0087052 1.31830.0001822 0.00055876 G₁₆T₁ 0.7661 0.00017384 0.00055876 1.20690.00045658 0.0010001 T₂₀ 0.60023 0.001288 0.0023699 1.2048 0.000577350.0012072 T₁G₁₆ 0.78642 0.0001312 0.00046425 1.0386 0.0020908 0.0036991CCATAATTCGAAACG 0.79837 0.0099977 0.01533 0.84121 0.010505 0.015588TTCTG T₁₆G₂ 0.60568 0.00075013 0.0015003 0.67964 0.044519 0.05851 G₇0.60019 0.0025561 0.0043548 0.58484 0.084445 0.10222 T₁₆G₁ 0.562830.00020274 0.00058287 0.58344 0.10765 0.12697 G₉ 0.76088 0.000309080.00083632 0.47427 0.18064 0.20774 (GT)₁₀ 0.14851 0.42349 0.45303-0.36072 0.26665 0.29917 C₂₀ 0.63319 0.04842 0.06187 -0.38242 0.283550.31056 (GA)₁₀ 0.78848 6.58E−05 0.00026755 0.079545 0.71587 0.74841(CG)₁₀ 0.5763 0.077347 0.096161 0.58108 0.78416 0.80158 G₁₄ 0.442260.013855 0.019917 -0.04129 0.88587 0.88587 ^(a)FDR, False discoveryrate.

Example 8 Antibody Reactivities to Synthetic Oligonucleotides in SLEPatients Compared to Those of Healthy Subjects

FIGS. 7A and 7B list the oligonucleotides with increased IgG (7A) andIgM (7B) binding in 77 SLE patients compared to 83 healthy controls. Abroad spectrum of oligonucleotides was found (7A) to be extremelySLE-indicative (p value ≤1.87E-08), sensitive (≥0.609), specific(≥0.769) and accurate (≥0.687).

Examples of such oligonucleotide antigens are G1T16 (SEQ ID NO: 42)having a p value 9.49E-15, sensitivity 0.680, specificity 0.822, andaccuracy 0.750, CCATAATTGCAAACGTTCTG (SEQ ID NO: 1) having a p value2.81E-12, sensitivity 0.657, specificity 0.798, and accuracy 0.725,G1T16G1 (SEQ ID NO: 5) having a p value 1.07E-18, sensitivity 0.767,specificity 0.869, and accuracy 0.819, CCATAATTGCAAAGCTTCTG (SEQ ID NO:3) having a p value 9.87E-14, sensitivity 0.659, specificity 0.830, andaccuracy 0.743, A20 (SEQ ID NO: 22) having a p value 1.87E-08,sensitivity 0.609, specificity 0.769, and accuracy 0.687, T20 (SEQ IDNO: 8) having a p value 8.00E-14, sensitivity 0.663, specificity 0.814,and accuracy 0.738, T16G2 (SEQ ID NO: 28) having a p value 4.81E-15,sensitivity 0.682, specificity 0.856, and accuracy 0.769, and T16G1 (SEQID NO: 7) having a p value 2.38E-11, sensitivity 0.656, specificity0.828, and accuracy 0.741.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

The invention claimed is:
 1. A method of determining the reactivity ofantibodies in a sample of a human subject suspected of having SystemicLupus Erythematosus (SLE), the method comprising: i. obtaining a serum,plasma, or blood sample from the human subject; ii. providing aplurality of oligonucleotide antigens immobilized on a solid support inthe form of an antigen probe set, an antigen array, or an antigen chip,said plurality of antigens comprising at least two oligonucleotideantigens selected from the group consisting of SEQ ID NOs: 42, 5, 28, 1,3, 7 and 22; iii. contacting the sample with the at least twooligonucleotide antigens of step ii., under conditions sufficient toform a specific antigen-antibody complex for each of the oligonucleotideantigens; and iv. quantifying the amount of antigen-antibody complex ofstep iii. formed for each oligonucleotide antigen, thereby determiningthe reactivity of the antibodies in the sample.
 2. The method of claim1, wherein the quantifying of step iv. is performed by a system thatallows quantitative measurement of antigen-antibody binding by laserscanning, light detecting, photon detecting via a photo-multiplier,photographing with a digital camera-based system or video system, orfluorescence detecting.
 3. The method of claim 1, wherein said pluralityof antigens comprises at least three of said oligonucleotide antigensselected from the group consisting of SEQ ID NOs: 42, 5, 28, 1, 3, 7,and 22 and said sample is contacted with the at least threeoligonucleotide antigens of step iii.
 4. The method of claim 1, whereinsaid plurality of antigens comprises at least four of saidoligonucleotide antigens selected from the group consisting of SEQ IDNOs: 42, 5, 28, 1, 3, 7 and 22 and said sample is contacted with the atleast four oligonucleotide antigens of step iii.
 5. The method of claim1, wherein the human subject is positive for dsDNA antibodies.
 6. Themethod of claim 1, wherein the human subject is negative for dsDNAantibodies.